1. Field of the Invention
This invention relates to a method for classifying leukocytes in the practice of clinical testing. More particularly, this invention relates to a method of classifying leukocytes with a flow cytometer by means of optical measurements on blood cells stained with fluorescent dyes, which method is capable of maintaining the desired precision of leukocyte classification irrespective of the difference in the lapse of time following blood sampling.
2. Prior Art
Leukocytes in the peripheral blood of normal subjects can be classified as being of five types consisting of lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Different leukocyte types have different functions and counting of leukocytes in the blood according to their type provides valuable information for diagnostic purposes. For instance, an increase in the number of neutrophils is-associated with inflammation, and diseases such as myocardial infarction and leukemia, and a decrease in their number is associated with vital diseases, hypoplastic anemia, agranulocytosis, etc. On the other hand, an increase in the number of eosinophils is found in such diseases as parasitosis, Hodgkin's disease and allergosis. An increased number of monocytes occurs either during the convalescence period of patients suffering from infectious diseases or in such diseases as monocytic leukemia.
Classification and counting of leukocytes have been made most commonly by the differential counting method which is also referred to as the visual counting method or simply as the manual method. In this method, a blood sample is smeared on a glass slide and the blood corpuscles in the smear are fixed and stained for examination by microscopy. The technician identifies the type of individual leukocytes according to their morphological features (e.g., their size, the morphology of their nucleus and cytoplasm, and the presence or absence of granules) or the degree of dye uptake and performs classification and counting of them. In ordinary laboratories, 100-200 leukocytes are usually counted for each sample and the percentage of the total leukocyte count occupied by each type of corpuscle is recorded as a measured value.
The differential counting method has several disadvantages. First, microscopic observation must be preceded by cumbersome procedures for preparing a specimen that involve such steps as smearing a blood sample on a glass slide, fixing the corpuscles and staining them. Secondly, it is a great burden for the technician to identify subtle differences between corpuscles by microscopic classification and counting. Thirdly, it is difficult even for a skilled technician to yield consistent counts by the manual method since aside from the small number of leukocytes computed, the smeared sample often has an uneven distribution of blood corpuscles.
Various methods have been proposed for eliminating these disadvantages of the manual method of leukocyte classification by achieving automation and such automated techniques may be roughly divided into two types. The first method consists of recording the images of corpuscles with a video camera or some other suitable imaging device and classifying the leukocytes by means of image processing on a computer. The operating principle of this method is similar to that of the conventional visual counting method but primarily due to the existence of many corpuscles that defy classification by processing with a computer, this method has not yet become an ideal alternative to the manual method. Furthermore, this method is not economically feasible since it requires sophisticated equipment which is large and costly.
The other approach toward automatic classification and counting of leukocytes is based on a flow system. In this method, a blood sample having corpuscles suspended in a diluent is permitted to flow in such a way that the corpuscles will individually (one by one) pass through a constricted detector and leukocyte classification is made by analyzing the signal generated by the detector. This second method of leukocyte counting which makes use of a flow system is further subdivided into two categories.
In a method of the first category, an electrolyte in which all red cells that were present have been destroyed with a lysing agent so that only leukocytes will be suspended is permitted to flow through an orifice and the change in electrical impedance that occurs at the orifice when each corpuscle passes through it is detected, with the magnitude of the detected signal being used as a basis for classification of leukocytes.
A method of the second category is characterized by the use of a flow cytometer that comprises a light source, a flow cell that permits the blood cells in a sample to flow one by one through a constricted channel, a photometric unit that detects light issuing from each blood cell, and an analyzer for analyzing the detected signal. In this method, the corpuscles in the sample which are stained are illuminated under light and the fluorescence emitted from the irradiated corpuscles is detected, optionally together with scattered light, with leukocyte classification being made in accordance with the intensity of the detected signal.
Techniques that fall within the category of this flow cytometric method are described in, for example, Japanese Patent Publication No. 853/1984 and L. A. Kamentsky, Blood Cells, 6, 121-140 (1980). According to these techniques, a blood sample is stained with 10 volumes of an Acridine Orange solution, incubated for 1 minute, and irradiated under a light source such as an argon ion laser. The green fluorescence and red fluorescence that are emitted from the individual corpuscles are measured and classification and counting of leukocytes are subsequently made based on a two-dimensional plot of the fluorescence measurements.
Other examples of techniques that are classified as being within the flow cytometric approach are shown in Unexamined Published Japanese Patent Application No. 20820/1975, H. M. Shapiro et al., J. Histochem. Cytochem., 24 (1) 396-411 (1976); and supra, 25 (8) 976-989 (1977). According to these methods, a blood sample is stained with 4 volumes of a Dye Solution I, incubated for 3 minutes, further mixed with 20% formaldehyde in a volume equal to the blood, fixed for 5 minutes, and diluted with a diluting Dye Solution II to obtain a concentration 15-20 times as low as the initial value. The so prepared specimen is subjected to measurement with a flow cytometer.
The flow cytometer employed in these methods uses either a mercury lamp that produces three different wavelengths of light or three lasers, so as to excite the three fluorescent stains in each of the dye solutions. The parameters measured are three kinds of fluorescence, forward scattered light, side-scattered light and absorbed light. Based on these six parameters, two-dimensional plots are constructed in four stages and analyzed to make leukocyte classification and counting.
Unexamined Published Japanese Patent Application No. 70166/1988 discloses a one-step staining process consisting of staining a blood sample with a dye solution comprised of a buffer solution, inorganic salts and fluorescent dyes. But this method has the problem that unlysed erythrocytes may adversely affect measurement data to produce unreliable results.
Unexamined Published Japanese Patent Application No. 134958/1988 discloses a two-step staining process that uses a hypotonic acidic fluorescent dye solution and a solution that will changes its osmolarity and pH. A blood sample stained with those solutions is loaded in a flow cytometer to obtain signals for fluorescence and side-scattered light and on the basis of those signals, a two-dimensional plot of fluorescence intensity vs the intensity of side-scattered light is constructed as shown in FIG. 2, whereby the leukocytes in the sample are classified into five types. Numerals 1-6 in FIG. 2 signify the populations of lymphocytes, monocytes, neutrophils, eosinophils, basophils and a noise component, respectively.
In the first version of the method that uses a flow system for leukocyte classification and counting, the disruption of erythrocytes is a prerequisite but depending on blood sample, it is impossible to effect complete lysis of erythrocytes and the accuracy of measurements may be impaired in such a case.
The examples of the flow cytometric approach that are described in Japanese Patent Publication No. 853/1984 and Blood Cells, 6, 121-140 (1980) are characterized by performing measurements before dye absorption by the cells reaches an equilibrium, or at the time when the difference between the intensities of fluorescence from individual leukocytes attains a maximum during the staining process. However, the time required for attaining an appropriate level of fluorescence intensity in a sample whose leukocyte count is at either one of two extremes will be different from the time for a normal sample and an appropriate staining time must be selected for each sample. As a further problem, this method relies solely on the differential intensity of fluorescence for leukocyte classification and does not necessarily ensure precise separation between different leukocyte types such as lymphocytes and monocytes.
The other examples of the cytometric approach that are described in Unexamined Published Japanese Patent Application No. 20820/1975, J. Histochem, Cytochem., 24 (1) 396-411 (1976) and supra, 25 (8) 976-989 (1977) have the disadvantage that they involve many steps of operation and staining takes a prolonged time and requires the use of reagents in a complex system. Furthermore, practice of these methods requires a very sophisticated and costly apparatus that includes three light sources and which is capable of measuring six parameters. In addition, analysis of such a large number of parameters is inevitably complicated and requires an analyzer having a large capacity.
All of the prior art techniques of flow cytometric approach including the method proposed by Unexamined Published Japanese Patent Application No. 70166/1988 have one common problem in that detection with a flow cytometer is impossible if neutrophils in a blood sample die when a long time passes before measurement. The method described in Unexamined Published Japanese Patent Application No. 134958/1988 aims at detecting dead cells to obtain correct neutrophil counts but, depending on the specimen to be assayed, it is sometimes impossible to achieve complete differentiation of dead cells. The problem also occurs in the case of damaged cells because the zone of their distribution becomes mixed up with the distribution zone of intact cells or another cell type, thereby making it impossible to perform complete differentiation between the two cell groups. In order to avoid these problems, measurements by the conventional flow cytometric methods always require the use of fresh blood samples.